This invention relates to the identification and isolation of DNA promoters from coffee. The invention also relates to a method of protein expression in transgenic plants.
Coffee is an agricultural commodity that plays a significant role in the economies of many developing countries. In Colombia, coffee cultivation is restricted to mountain areas with altitudes between 1200 and 1400 meters above sea level. It is especially concentrated in the central region, in an area called the Coffee Zone. With a total annual yield of around 12 million bags, Colombia is ranked second in world production. Of this production, 26% is used for domestic consumption, and the rest is exported to Europe (6 million bags), United States (3 million bags), and Asia (1 million bags), with an average annual market value (1991 to 1995) of $1.6 billion (Banco de la Republica, Indicadores Economicos NI 828 Banco de la Republica, Bogota, Colombia (1996)).
The genus Coffea belongs to the Rubiaceae family which includes other important plants, such as ipecacuanha (Cephaelis ipecacuanha) and cinchona (Cinchona spp.). The genus contains about 70 species, most of them trees and shrubs growing at low altitudes in the tropical rain forests of Africa and Asia (Sondahl et al., xe2x80x9cCoffee,xe2x80x9d Biotechnology of Perennial Crops CAB International, Wallingford, UK (1992)). Only two species are widely cultivated, Coffea arabica and Coffea canephora. All known species are diploid (2n=2X=22 chromosomes) and obligate outbreeders with self-incompatibility systems, except for C. arabica which is tetraploid (2n=4X) and self-fertile.
The species Coffea arabica L probably originates from a relatively recent cross between C. eugenoides and C. canephora, a hypothesis supported by random amplified polymorphic DNA""s (RAPD) (Lashermes et al., xe2x80x9cUse of Amplified DNA Markers to Analyze Genetic Variability and Relationships of Coffea Species,xe2x80x9d Genetic Resources and Crop Evolution 40:91-99 (1993)) and chloroplast restriction fragment length polymorphism (RFLP) analyses (Lashermes et al., xe2x80x9cInheritance and Restriction Fragment Length Polymorphism of Chloroplast DNA in the Genus Coffea L.,xe2x80x9d Theoretical and Applied Genetics 93:626-632 (1996)). The nuclear DNA content of C. arabica, as determined by flow cytometry, is 2.4 pg/interphase nucleus, or n=2X=1158 Mb (Arumuganathan et al., xe2x80x9cNuclear DNA Content of Some Important Plant Species,xe2x80x9d Plant Molecular Biology Reporter 9:208-218 (1991)). It is cultivated in 75% of the coffee plantations around the world. The quality of the beverage is potentially excellent, being known in the trade as xe2x80x9cmild coffee.xe2x80x9d The most important pests affecting this species are coffee rust (Hemileia vastatrix), coffee berry disease (CBD, Colletotrichum coffeanum), and coffee berry borer (Hypothenemus hampei, Coleoptera). Worldwide, these three pests cause an estimated crop loss of 14.8%, or about $1 billion annually (Oerke et al., xe2x80x9cEstimated Losses in Major Food and Cash Crops,xe2x80x9d Crop Production and Crop Protection, Elsevier, New York, U.S.A. (1994)).
Several cultivars have been described for C. arabica, but because of the narrow genetic base of the species, they are due mainly to single gene mutations. The commonly grown varieties, Tipica and Bourbon, can grow up to 6 m tall under natural conditions. Coffee trees grow well at tropical elevations, ranging from 300 to 1200 m above sea level, with a mean annual temperature of 18 to 21xc2x0 C. C. arabica cv. Caturra is a mutant of the Bourbon cultivar, which was discovered in Brazil in 1949 and has been extensively grown in Colombia. The main characteristic of this cultivar is the dwarf phenotype resulting from the action of a dominant gene that reduces the internode distance (Orozco, xe2x80x9cDescripcion de Especies y Variedades de Cafxc3xa9,xe2x80x9d CENICAFE Chinchinxc3xa1, Caldas (1986)). Use of this phenotype has allowed planting densities to increase from 2500 plants to 10,000 plants/h, which, in turn, has increased bean yields from 5000 kg to about 8000 kg/h.
The species Coffea canephora Pierre ex Froehner, also known as Coffea robusta Linden, is the diploid species most widely cultivated around the world. It is self sterile and cross pollinated and therefore much more variable than C. arabica. C. canephora is better adapted to humid-hot climates and is frequently cultivated in low to medium altitudes. The quality of the beverage made from C. canephora is usually regarded as inferior to that made of C. arabica. However, C. canephora is more resistant to coffee rust and CBD.
Traditionally, Tipica, Bourbon, and Caturra were the C. arabica cultivars grown in Colombia. These varieties produce a high quality coffee, but they are very susceptible to pests which are not held in balance by natural biocontrol. Although South America was free of the most important coffee pests for many years, threats became real with the appearance of coffee rust in Brazil in 1970 and in Nicaragua in 1976. This disease finally arrived in Colombia in 1983. In anticipation, the Colombian National Center of Coffee Research (CENICAFE), a organization of Colombian coffee growers, began a breeding program for resistance to coffee rust in 1968. The purpose was to create a cultivar of C. arabica that preserves the traditional cup quality, but incorporates increased genetic diversity, durable resistance to the coffee rust, phenotypic homogeneity, and productivity (Castillo et al., xe2x80x9cLa Variedad Colombia: Seleccixc3x3n de un Cultivar Compuesto Resistente a la Roya del Cafeto,xe2x80x9d CENICAFE Chinchinxc3xa1, Colombia, 171 p. (1986)).
Timor hybrid was chosen as the resistant parent of the new cultivar since no germplasm of C. arabica was known to contain durable resistance genes against coffee rust. Timor hybrid is a natural interspecific hybrid between C. arabica and C. canephora found in 1917 on the island of Timor, Indonesia. Used in Africa and India for many years, it showed broad resistance against the local rust races. In the Colombian breeding program, the recurrent quality parent of the new cultivar was C. arabica cv. Caturra, which in addition to providing the characteristics of dwarfism and good beverage quality, was a familiar cultivar among the growers. The result was the release in 1980 of the Colombia cultivar, a composite cultivar made up by the mixture of seeds coming from the best F5 and F6 progenies resistant to coffee rust and with optimal adaptation to the climate and soils of the Colombian coffee zone (Castillo et al., xe2x80x9cLa Variedad Colombia: Seleccixc3x3n de un Cultivar Compuesto Resistente a la Roya del Cafeto,xe2x80x9d CENICAFE Chinchinxc3xa1, Colombia, 171 p. (1986)).
Components of the Colombia cultivar are continuously tested for their resistance against coffee rust and other diseases. When a component is found susceptible, it is withdrawn from the mixture. In the same way, new selected components can be added to the cultivar. This procedure provides a dynamic update of the cultivar in its resistance against coffee rust. Seed production and distribution of the Colombia cultivar are carried out exclusively by the National Federation of Coffee Growers. This maintains a diversity in resistance to coffee rust as well as the phenotypic homogeneity, yet results in low seed prices for the farmers.
In contrast to many other crops, coffee has not been the subject of extensive research in molecular biology. This may be due to factors such as the long life cycle, the difficulty of maintaining plants out of the tropical environment, and the lack of resources from countries that cultivate coffee. Nevertheless, some advances are being made in this field.
Several proteins, especially those involved in the resistant interaction with coffee rust have been studied. Kinetics and differential expression of phenylalanine ammonia lyase (PAL) (Almario, xe2x80x9cStudy of the Activity of the Phenylalanine Ammonia Lyase in the Presence of the Pathogen in Coffee Varieties Resistant and Susceptible to Hemileia vastatrix Ber and Br.,xe2x80x9d Universidad Nacional de Colombia, Bogota, 155 p. (1992)), superoxide dismutase (Daza et al., xe2x80x9cIsoenzyme Pattern of Superoxide Dismutase in Coffee Leaves from Cultivars Susceptible and Resistant to the Rust Hemileia-vastatrix,xe2x80x9d Journal of Plant Physiology 141:521-526 (1993)) and lipoxygenase (Rojas et al., xe2x80x9cStimulation of Lipoxygenase Activity by Cotyledonary Leaves of Coffee Reacting Hypersensitively to the Coffee Leaf Rust,xe2x80x9d Physiological and Molecular Plant Pathology 43:209-219 (1993)) of the Caturra and Colombia cultivars have been compared.
Phenylalanine ammonia lyase (PAL) is a key enzyme that catalyzes the deamination of L-phenylalanine to produce cinnamic acid. Cinnamic acid is a substrate that feeds several biosynthetic routes, leading to the production of various classes of phenylpropanoid-derived secondary plant products. Some of these products are involved in aspects of the normal development of the plant such as petal pigmentation and xylem development. However, many of them are directly involved in the plant defense response (Hahlbrock et al., xe2x80x9cPhysiology and Molecular Biology of the Phenylpropanoid Metabolism,xe2x80x9d Annual Review of Plant Physiology and Plant Molecular Biology 40:347-369 (1989)).
Activation of PAL can lead to the accumulation of lignin, suberins, and a variety of phenolic esters that increase the strength of cell walls. (Hammond-Kosack et al., xe2x80x9cResistance Gene-Dependent Plant Defense Responses,xe2x80x9d The Plant Cell 8:1773-1791 (1996)). Also, PAL is necessary for the synthesis of flavonoid derivatives that function as pigments, as well as in intracellular signaling, UV protectants, phytoalexins and coumarins, and salicylic acid. PAL is also involved in the synthesis of acetosyrnigone, a wound metabolite that serves as a signal for the activation of virulence (vir)genes in Agrobacterium tumefaciens. 
PAL is usually encoded by a small gene family of 2 to 6 members. In some plants (e.g., Solanum tuberosum), 40 PAL genes can be detected (Joos et al., xe2x80x9cPhenylalanine Ammonia Lyase in Potato (Solanum tuberosum L.) Genomic, Complexity, Structural Comparison of Two Selected Clones and Modes Of Expression,xe2x80x9d European Journal of Biochemistry 204:621-629 (1992)), while in loblolly pine (Pinnus taeda), there seems to be only one (Whetten et al., xe2x80x9cPhenylalanine Ammonia Lyase from Loblolly Pine: Purification of the Enzyme and Isolation of Complementary DNA Clones,xe2x80x9d Plant Physiology 98:380-386 (1992)). It is supposed that different members of the family respond differentially to the induction signals, either in their kinetics of induction, or accumulation of transcripts.
PAL activity is mainly regulated at the level of transcription, by the synthesis of new mRNA (Lois et al., xe2x80x9cA Phenylalanine Ammonia-Lyase Gene from Parsley: Structure, Regulation and Identification of Elicitor and Light Responsive Cis-Acting Elements,xe2x80x9d EMBO Journal 8:1641-1648 (1989)). Perturbation of the normal PAL expression in transgenic plants generates abnormal development phenotypes (Elkind et al., xe2x80x9cAbnormal Plant Development and Down-Regulation of Phenylpropanoid Biosynthesis in Transgenic Tobacco Containing Heterologous Phenylalanine Amrnonia-Lyase Gene,xe2x80x9d Proceedings of the National Academy of Sciences of the USA 87:9057-9061 (1990)). Accumulation of PAL transcripts has been observed in the presence of developmental cues, wounding (Ohl et al., xe2x80x9cFunctional Properties of a Phenylalanine Ammonia-Lyase Promoter from Arabidopsis,xe2x80x9d The Plant Cell 2:837-848 (1990)), hypersensitive response (Dong et al., xe2x80x9cInduction of Arabidopsis Defense Genes by Virulent and Avirulent Pseudomonas syringae Strains and by a Cloned Avirulence Gene,xe2x80x9d The Plant Cell 3:61-72 (1991)), ozone fumigation (Sharma et al., xe2x80x9cOzone induced Expression of Stress Related Genes in Arabidopsis Thaliana,xe2x80x9d Plant Physiology 105:1089-1096 (1994)), and insect saliva (Hartley et al., xe2x80x9cBiochemical Aspects and Significance of the Rapidly Induced Accumulation of Phenolics in Birch Foliage,xe2x80x9d Tallamy, ed., Phytochemical Induction in Herbivores, New York: John Wiley (1991)).
Every cell in the plant has to fulfill basic needs to survive. For this purpose, a set of proteins is constantly present in the cell which is involved in functions such as membrane traffic, membrane stability, cytoplasm organization, transcription apparatus, and primary metabolic pathways. Genes encoding these proteins are called xe2x80x9chousekeeping genes,xe2x80x9d and they are controlled by promoters that are active almost permanently during the cell cycle. Such promoters are called xe2x80x9cconstitutive.xe2x80x9d Since the default state of eukaryotic promoters is xe2x80x9coffxe2x80x9d (contrary to prokaryotic promoters; Lewin, xe2x80x9cGenes V.,xe2x80x9d Oxford University Press, Oxford, UK (1994)), constitutive promoters must contain structural features that enable them to remain active in several tissues and during the multiple developmental stages of the plant. By sequencing, comparing, and modifying plant promoters, it has been possible to identify functional components in their DNA sequence.
In contrast to housekeeping genes, some genes encode products that are only required under special conditions related to developmental stages of the plant, environmental stress, or pathogen attack. These genes contain xe2x80x9cinducible promotersxe2x80x9d that can be turned on quickly by an inducer agent and are active for a limited length of time before they are turned off again. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent, such as a virus or fungus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen. In addition, inducible promoters include promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue specific promoters include seed, flower, or root specific promoters as are well known in the field (see e.g., U.S. Pat. No. 5,750,385 to Shewmaker et al.). For transgenic plants, these promoters are of interest if accumulation of the protein product, for biological or marketing reasons, is desired in certain tissues, or at certain times.
Genetic engineering provides valuable tools for studying promoter activity. By making constructs in which a reporter gene is fused under the control of a promoter sequence, it is possible to observe the specific activity of the promoter by monitoring the expression of the reporter gene (Herrera-Estrella et al., xe2x80x9cChimeric Genes as Dominant Selectable Markers in Plant Cells,xe2x80x9d EMBO Journal 2:987-995 (1983)). Gene fusion not only provides a way to eliminate variables associated with post-transcriptional regulation from the experiment but also allows comparisons among different promoters or among variations of the same promoter (promoter deletion analysis).
Although a powerful tool in the study of gene control, gene fusion sometimes requires additional analyses in order to provide meaningful results. This is especially true in the characterization of tissue specific promoters. In this case, sequences containing the information for tissue specificity are not only present in the 5xe2x80x2 upstream region of the gene, but can also occur in the downstream coding region or even in the 3xe2x80x2 end region (Fu et al., xe2x80x9cHigh-Level Tuber Expression and Sucrose Inducibility of a Potato Sus4 Sucrose Synthase Gene Require 5xe2x80x2 and 3xe2x80x2 Flanking Sequences and the Leader Intron,xe2x80x9d Plant Cell 7:1387-1394 (1995); and Sieburth et al., xe2x80x9cMolecular Dissection of the Agamous Control Region Shows that Cis Elements for Spatial Regulation are Located Intragenically,xe2x80x9d The Plant Cell 9:355-365 (1997)).
So far, the Colombia cultivar has remained resistant to coffee rust and other coffee pathogens in Colombia. It is possible, nonetheless, that new strains of Hemileia vastatrix could be selected for virulence or that new diseases could be introduced to the hemisphere that are pathogenic to the Colombia cultivar. For these reasons, components of the Colombia variety are permanently being screened for resistance against important pathogens, and, taking advantage of the dynamic nature of a multiline, components are continually added to or removed from the final product delivered to the farmers.
Furthermore, there is no known resistance to the coffee berry borer, Hypothenemus hampei, at the cultivar or species levels, and, at the time the borer appeared in Colombia (1988), no resistant material had been introduced into the Colombia cultivar. The main obstacle to developing cultivars resistant to coffee berry borer is the lack of resistant parents. Pathogens create a constant selective pressure on the plant population, which has resulted in the co-evolution of the mechanisms of attack and defense (Agrios, xe2x80x9cPlant Pathology, 4th Ed.,xe2x80x9d Academic Press, San Diego, U.S.A., 635p (1997)). The natural conditions in which this process develops are altered by the agricultural practice of monoculture which has resulted in enormous crop losses caused by pathogens that have evolved while the host has been held stable.
Because of the low variability present in natural populations of C. arabica, resistance must be sought in other species. Even if a related species is found to be resistant, transfer of resistance genes from a wild relative of coffee into the cultivated varieties of C. arabica can be difficult or impossible due to interspecific incompatibility and the diploid-tetraploid nature of the cross. Despite that, by use of tissue culture for embryo rescue, crosses between species that are very distant from each other in the phylogenetic tree can be enabled; however, the long term backcrossing program needed to select away from characters introgressed from the wild species makes this a difficult option.
The present invention relates to an isolated DNA promoter suitable for inducing expression of a protein encoded by a second DNA operably associated with the DNA promoter. The DNA promoter of the present invention is isolated from coffee.
The present invention also relates to a DNA construct. The DNA construct has a DNA promoter isolated from coffee, operably linked 5xe2x80x2 to a second DNA. The second DNA encodes for a protein or polypeptide, so that transcription of the second DNA is induced. The construct also contains a 3xe2x80x2 regulatory region operably linked to the second DNA.
The present invention also relates to transgenic plants and seeds produced by transformation with the DNA construct containing a DNA promoter operably linked to a second DNA encoding a protein or polypeptide.
The present invention also relates to a method of directing protein expression in plants. This involves transforming a plant cell with a DNA construct of the present invention. This construct contains a DNA promoter isolated from coffee that is associated with a second DNA encoding a protein or polypeptide. The DNA promoter induces expression of the second DNA in a plant regenerated from the transformed plant cell.
The present invention also relates to transgenic plants and seeds produced by transformation with the DNA construct containing the DNA promoter isolated from coffee and a DNA encoding a protein.
The present invention also relates to a method of directing protein expression in plants. This involves transforming a plant cell with a DNA construct containing a DNA promoter of the present invention and regenerating a plant from the transformed plant cell. The expression of the second DNA of the DNA construct, under control of the DNA promoter, then occurs in the transformed plant.
The low gene diversity of Coffea spp., the lack of resistance to coffee plant pests such the coffee berry borer, and the potential for widespread coffee crop devastation due to pathogen infestation combine to present a serious need for improved methods of coffee plant disease control. In the short term, the options for the farmers are cultural, chemical and biological control. In the long term, the solution is the incorporation of resistance into the Colombia cultivar. With the development of an efficient transformation system, new genes could be introduced into different components of the coffee cultivar, without altering other significant traits of the plants. This option could translate into a reduction of production costs for the farmer, as well as a reduction in pesticide residues on the product and in the environment.
In contrast to sexual breeding, biotechnology can provide a rapid way to transfer genes into coffee in order to improve cultivars that have already been subjected to an extensive selection process for critical factors, such as production, cup quality, and coffee rust resistance.
The present invention provides an important tool for the expression of DNA molecules of choice in plants. This will increase the potential for development of pathogen resistant cultivars of coffee, and improve other characteristics of coffee plants, such as hardiness, production, and cup quality, while overcoming the deficiencies of the methods currently available for fighting disease in the coffee plant.